Embodiments of the invention are directed to the field of optical systems, particularly to objective lenses and, more particularly to objective lenses for use in fluorescence collection and imaging in multiphoton (MP) and second harmonic generation (SHG) applications including microscopy and endoscopy.
The multiphoton imaging techniques known as multiphoton microscopy (MPM) has developed into a standard tool for the life scientist with far reaching applications ranging from basic cell biology to imaging physiology and disease progression in live animals. This robust form of laser scanning microcopy is ideal for experiments in which cellular and sub-cellular resolution fluorescence imaging is required in a highly scattering medium. As the technique has advanced there has been a corresponding development of new objective lenses specifically for use in multiphoton microscopy. These new designs have increased IR transmission and obtained high numerical apertures (NAs) at relatively low magnification (e.g., Olympus 25×/1.05 NA; Zeiss 20×/1.0 NA). Other collection schemes have been reported, which exploit the principle that all of the collected emission light in MPM—even the scattered photons—contribute to useable signal, have increased the overall solid angle sampled by implementing additional collection optics such as a parabolic reflector underneath the tissue, or a ring of waveguides surrounding it. High NA reflecting objectives that efficiently collect fluorescence have also been demonstrated.
Although high numerical aperture is important to achieve the highest resolution, there are cases in which the experimenter is willing to sacrifice resolution for an increase in the field of view (FOV), for example, in cell tracking experiments in tissues or for observations of calcium oscillations in large neuronal networks. Additionally, a wider field of view would be particularly useful for “multiphoton pathology” applications. Existing technology has been focused on higher numerical aperture objective designs with fields of view under a millimeter. The translation of MPM from the laboratory to the operating room is currently underway with various research groups developing MPM endoscopes capable of performing in-vivo optical biopsies. The direct MPM imaging of fresh unstained biopsy tissue has been demonstrated to provide instant histological grade images without the tedious preparation necessary to produce standard sectioned slides, and may become an indispensable part of clinical practice. In addition, the intrinsic contrast provided both by autofluorescence from endogenous fluorophores such as NADH, flavins and other autofluors, as well as second harmonic generation from such macromolecules as collagen, adds information beyond morphometric parameters. However, some of these signals are weaker than typical dyes by several orders of magnitude, and it is therefore important to maximize the collection optics for this particular application.
In view of the foregoing discussion and the current state of technology represented here, the inventors have recognized a need for new objective lens designs motivated by the requirements of: (1) large field of view; (2) long working distance; (3) a sufficient numerical aperture for reasonable optical sectioning and single cell resolution; (4) collection of as large of a solid angle as possible; (5) an inexpensive design to fabricate; and (6) chromatic correction over at least the bandwidth of a typical femtosecond pulse (10-15 nm), as well as advantageous and beneficial solutions thereto as provided according to embodiments of the invention as disclosed below.